The main goal of our research is to understand the structure and function of plasmodesmata (PD), cytoplasmic channels that span cell walls to adjacent plant cells. As plant cell fate is determined by positional information, PD provide a means whereby cells can coordinate differentiation, transporting short- or long-range signals. With their critical roles established, it is now essential to reveal the molecular players involved in PD activity. We hypothesize that specific gene products regulate the structure and function of these dynamic channels. Indeed, we determined that PD are regulated during 3 distinct stages of development, leaf maturation, the induction of flowering, and mid-embryogenesis. We developed a direct bioassay for PD function during embryogenesis in Arabidopsis to identify temporal and spatially restricted changes in PD function. Embryos are easily accessible, and contain few cells that undergo profound developmental changes. We propose to exploit Arabidopsis embryogenesis as an advantageous model system to: 1. Perform a detailed study of cell-to-cell trafficking during embryogenesis. As we have shown PD are down-regulated during mid-embryogenesis, we propose to test for additional times when PD alter their aperture. We predict cell-to-cell transport will be high in regions of new growth and development, and decrease as embryos mature. We will determine the aperture of PD during different stages, to predict the size of developmental factors transported during embryogenesis. We will analyze the kinetics of transitions in PD function; shifts in permeability predict changes in gene expression of PD components or regulators. 2. Identify and characterize mutant lines affecting the structure and function and regulation of PD. This aim builds on Aim 1, as determination of developmental transitions provides entry points to identify mutants with altered cell-to-cell transport, and genes that affect PD function. Alterations of PD likely have severe defects in growth; thus, PD mutants are expected to manifest first as defects during embryo development. Fifteen mutant lines with altered cell-to-cell transport were uncovered. We will prioritize the study (morphology, gene mapping, cloning, localization, functional tests) of 4 lines, three with increased cell-to-cell transport of macromolecules and one line with decreased traffic of small tracers. 3. Analyze how a specific gene product affects the structure or regulation of PD. We have recently discovered the gene interrupted in one mutant line with increased PD aperture. We will ask how this gene product affects the structure or regulation of PD by determining a) its cell localization, b) whether it acts directly or indirectly in PD transport using different functional assays we have developed, and c) interacting partners (such as protein co-factors) that are essential for its function in cell-to-cell transport.